Parental competition for the regulators of chromatin dynamics in mouse zygotes

The underlying mechanism for parental asymmetric chromatin dynamics is still unclear. To reveal this, we investigate chromatin dynamics in parthenogenetic, androgenic, and several types of male germ cells-fertilized zygotes. Here we illustrate that parental conflicting role mediates the regulation of chromatin dynamics. Sperm reduces chromatin dynamics in both parental pronuclei (PNs). During spermiogenesis, male germ cells acquire this reducing ability and its resistance. On the other hand, oocytes can increase chromatin dynamics. Notably, the oocytes-derived chromatin dynamics enhancing ability is dominant for the sperm-derived opposing one. This maternal enhancing ability is competed between parental pronuclei. Delayed fertilization timing is critical for this competition and compromises parental asymmetric chromatin dynamics and zygotic transcription. Together, parental competition for the maternal factor enhancing chromatin dynamics is a determinant to establish parental asymmetry, and paternal repressive effects have supporting roles to enhance asymmetry.

Additional comments: -Because all experiments presented in this work are based on FRAP, the authors should provide sufficient details of their FRAP experiments and analysis in the Method. Please do not use "performed as described previously". I had a hard time finding the authors' previous paper that described the formula for the calculation of the mobile fraction. -line 280: I don't think the data that the authors presented are sufficient to support this model. Additional experiments (e.g., CHX) are needed to make this claim. Otherwise, the authors should tone down the discussion. -It is my understanding that the term "chromatin relaxation" refers to a response to DNA damage, which I think is not what the authors are studying in this manuscript. The authors may use a different term. -line 218: ""Sr" indicates strontium." should be in the legend of (A), not (E)?
Reviewer #2 (Remarks to the Author): There are several interesting phenomena described in Ooga, et al. manuscript. The major problem with the paper is the lack of mechanistic understanding of the relaxation vs compaction properties of pronuclei and what biological significance either of those states impart into the zygote.
There is a high degree of technical expertise, the micromaninpulations required in order to perform these experiments is incredibly impressive.
Since most of the experiments were done in the early to mid zygote stage before the first S-phase, which begins around 9 hours post-fertilization (PMID: 20442707) and the subsequent S phase will replace 50% of the histones that package each pronucleus, it is unclear whether the compaction status of the pronculei, or the differentiation compaction status comparing the mat-PN vs pat-PN, has any functional significance for the embryo. For instance, does the compaction status affect the timing to the first mitosis, the minor wave of ZGA that occurs in the late zygote preferentially from the paternal pronucleus, or the major wave of ZGA that is executed in the 2-cell stage of the mouse embryo? The authors have established a system in which to perturb the compaction status (ICSI with 2 pat-PNs, parthenotes, etc) but they have not fully capitalized on this technical expertise to answer a meaningful biological function.
I think some of their claims are well-supported such as the effect seen in Fig 1h (and also described in lines 110-112), in which presumably the multiple pat-PNs decrease the FRAP recovery of the chromatin in both the pat-PN and the mat-PN, and this is a dose-dependent response--but no comment is made on the molecular mechanism. Is this just a simple titration of the H3.3/HIRA stores that presumably are responsible for FRAP recovery before the first S-phase and replicative histones are deposited?
There is also significant experimental detail lacking in the text and the figure  The authors use the terms "compaction" and "relaxation" but what they really are measuring is mobility, ie the deposition of non-bleached (newly translated?) histone into the chromatin. Both compaction/relaxation are related to mobility, for instance non-replicative histones are often deposited into sites of high histone turnover (PMID: 20508129), which by necessity need to be accessible to chaperones or remodelers, but the terms should not be used interchangeably. If the authors want to measure accessibility, there are several methods that non-epigenomicists use such as this imaging-based MNAse assay (PMID: 28846101).
Reviewer #3 (Remarks to the Author): Ooga et al investigate chromatin dynamics in zygotes, using the mouse model. Studying chromatin dynamics in early embryogenesis is important because chromatin states have significant impacts on embryo development through modulating the transcription activity. The authors have used their previously established method to quantify chromatin dynamics in paternal and maternal pronuclei (PN) and found that paternal PN brings the chromatin compaction activity to the zygote, while the oocyte cytoplasm harbors the opposing chromatin relaxation activity. The data presented in the manuscript is solid and support most of their main conclusions. However, the manuscript would greatly benefit from additional experiments and text editing. It is striking to me that the parental asymmetry in chromatin dynamics was completely flipped when the timing of the sperm injection was delayed (Fig 2E-G). This point should be one of the main conclusions, as changing the fertilization timing was sufficient to reverse the parental asymmetry regardless of the paternal PN's compaction factors. Based on this result, I think the primary mechanism underlying parental asymmetry is the paternal PN having the advantage to start incorporating the relaxation factors first. And paternal PN's compaction factors have supporting roles to enhance this asymmetry. I recommend the authors to reorganize the manuscript to highlight this point. My another major point is how do the authors distinguish if it is the effect of the compaction factors or the competition for the relaxation factors that is compacting chromatin. This point was not clear to this reviewer in this manuscript. For example in Fig 2A and B, the chromatin of maternal PN became more compact when there are more maternal PN. The authors concluded that this is due to the competition for the relaxation factors between maternal PN. However, it is also possible that maternal PN (or maternal chromosomes they transferred) has compaction factors, and this is what making maternal PN more compact when there are more of them (like the experiments in Fig 1G and H where they increased the number of paternal PN). I strongly recommend the authors to clarify what makes the authors conclude if it is the effect of compaction factors or the competition for the relaxation factors that is making chromatin more compact in each experiment. Otherwise, this reviewer thinks that there are multiple other possible models to explain the data.
Additional points -line 162: brief explanation of zFRAP should come up much earlier in the Results section when it is first used. -line 164: This is not a specialized journal, and the authors should clearly explain the differences between ROSI, ELSI, tICSI, ICSI, and iICSI, so that the readers would understand why the authors need to perform these experiments to test the hypothesis.
-line 245: The subtitle "More chromatin relaxer was utilized in sp-mPN than fPN" is an overstatement, because what the authors have performed is treating zygotes with different RNA polymerase inhibitors and not directly working with the relaxation factors.
-line 257: To directly test the idea that the relaxation factors are supplied through the zygotic translation, it would be interesting to treat zygotes with Cycloheximide to block translation. In this manuscript, Ooga et al study eGFP-H2B dynamics as a proxy for chromatin structure in 5 mouse zygotes. The authors have previously observed that sperm male pronuclei (mPN) and 6 female pronuclei (fPN) show distinct dynamics of eGFP-H2B recovery after photobleaching. In 7 the present study, the authors show that sperm plays a role in the distinct FRAP dynamics 8 between mPN and fPN. Using vigorous oocyte manipulation techniques that allow them to 9 study effects of the number of PNs as well as their origin (mPN or fPN) on eGFP-H2B 10 dynamics, the authors propose that PNs compete for "chromatin relaxation factors", resulting in 11 different chromatin states between PNs. I think the idea of the competition model is interesting; 12 however, I have a major concern about the interpretation of FRAP data, which would impact the 13 main conclusion of the manuscript. F_post)) as a proxy for "chromatin relaxation". However, the values of MF may be affected by 17 the presence of free eGFP-H2B molecules in the nucleus. Indeed, the timescale of the recovery 18 that the authors observe is the order of seconds, which is rather consistent with the diffusion of 19 freely diffusing molecules than the slow diffusion of chromatin (e.g., Chalut et al., Biophysical 20 Journal, 2012). In normal somatic cells, free histones get rapidly degraded. However, oocytes 21 and early embryos of many species are provisioned with maternal histone pools and excess free 22 histones likely accumulate in the nucleus on top of the chromatin-bound histones. Furthermore, 23 the authors use eGFP-H2B mRNA injection, which is technically overexpression. My concern 24 is that the differences in MF may be due to differences in the pool size of free eGFP-H2B and 25 may not represent changes in the chromatin structure. For instance, in  Response: Thank you for your valuable suggestion. As per your suggestion, the amount 33 of free histone may affect histone mobility. We have recognized this importance. Therefore, in 34 our previous study, we confirmed the scarce free histone in the PN with detergent-treated zygotes 35 (Confidential Figure for reviewer only 1, see below) (Ooga et al., 2016). Prior to fixation, the 36 zygotes were treated with Triton X-100, owing to this the unbound protein fraction to chromatin 37 was washed away (Hajkova et al. 2010). In our results, Triton X-100 treatment did not decrease 38 the amount of eGFP-H2B but eGFP protein used as control. Therefore, there is no/negligible free 39 eGFP-H2B in the pronucleus of our experimental condition. To clarify this, we have added the 40 sentence below in the materials and methods section. "Notably, 1PN ICSI and 1PN-partheno have similar sized PN, but they showed distinct 67 histone mobilities (Fig. 1e, f). Furthermore, 1♂ (2sp) and 2♂ (2sp) zygotes whose PN sizes were 68 different, showed similar histone mobility levels (Fig. 1g, h), indicating that histone mobility was 69 determined regardless of PN size." Following your suggestion, we examined the eGFP-H2B amounts and found that there was no 80 significant difference in the male PN of both types of zygotes. Therefore, we added the sentence 81 below in the Results section. were set at 40 × 40 pixels (7.6 µm 2 ). Prior to bleaching, 3 pictures were taken at 1.6-s intervals. Response: Thank you for your suggestion. CHX use is ideal to prove that the 138 enhancing ability depends on zygotic translation. However, CHX also inhibits eGFP-H2B 139 translation from injected mRNA, which is required for our zFRAP experiment (Confidential 140 Figure only for reviewers 3; see below). Therefore, it is impossible to investigate histone 141 mobility in CHX-treated zygotes and we changed the results section and its interpretation. block not only Pol I but also DNA polymerase, but in our experiment DNA replication was not 147 affected (Supplementary Figure 13). Both inhibitors induced the reduction of histone mobility 148 particularly in sp-mPN, suggesting that pol I and pol III mediated transcription is involved in the 149 higher histone mobility in sp-mPN (Supplementary Figure 14 a-d)." parental asymmetric transcriptional activity. We added the data (Fig. 3) possibly due to a more transcriptional permissive chromatin state than fPN (Schultz 2002). 212 Therefore, we hypothesized that the higher histone mobility in sp-mPN is important for 213 regulating parental asymmetric transcriptional activities. To examine this, we took an advantage 214 of our discovery that delay-ICSI induced the reversed parental asymmetric chromatin dynamics 215 (Fig. 2e, f, and g). As expected, the longer delay reduced transcriptional activity in sp-mPN, 216 and eventually reversed parental asymmetric transcription was observed (Fig. 3a, b). 217 Considering that eviction and deposition of histone H2B could be coupled with transcription, it 218 is possible that lowered transcriptional activity caused slower histone mobility. Nevertheless, 219 the treatment with alpha-amanitin (ama), which is a RNA-polymerase II (Pol II) inhibitor, led to 220 no reduction of histone mobility (Fig. 3c, d). These results suggest that chromatin dynamics are 221 involved in the regulation of the pol II-mediated parental asymmetric transcriptional activity."  block not only Pol I but also DNA polymerase; however, in our experiment, DNA replication was 262 not affected (Supplementary Figure 13). Both inhibitors induced the reduction of histone 263 mobility particularly in sp-mPN, suggesting that pol I and pol III mediated transcription is 264 specifically involved in the higher histone mobility in sp-mPN (Supplementary Figure 14 a-d) reviewers' comment, we changed "chromatin relaxation" into "chromatin dynamics" or "histone 280 mobility" throughout the paper depending on the context. Both chromatin dynamics" and "histone 281 mobility" were used in the previous studies with FRAP that were performed by other research 282 groups (Bošković et al. 2014). It is striking to me that the parental asymmetry in chromatin dynamics was completely flipped 295 when the timing of the sperm injection was delayed (Fig 2E-G). This point should be one of the 296 main conclusions, as changing the fertilization timing was sufficient to reverse the parental 297 asymmetry regardless of the paternal PN's compaction factors. Based on this result, I think the 298 primary mechanism underlying parental asymmetry is the paternal PN having the advantage to 299 start incorporating the relaxation factors first. And paternal PN's compaction factors have 300 supporting roles to enhance this asymmetry. I recommend the authors to reorganize the 301 manuscript to highlight this point. "Notably, this competition is critical to the establishment of parental asymmetric 342 chromatin dynamics (Fig. 2e-g) and is involved in the regulation of parental asymmetric 343 transcriptional activity (Fig. 3a-d)." 344 (p22 line 354-359) 345 "Additionally, changing the fertilization timing was sufficient to reverse the parental 346 asymmetry regardless of the paternal repressive effects on chromatin dynamics suggest that 347 primary mechanism underlying parental asymmetry is the paternal PN having the advantage to 348 start incorporating the chromatin dynamics promoting factors first. And paternal repressive 349 effects have supporting roles to enhance this asymmetry (Fig. 3e)."  Figure 5). Furthermore, MF of 2♂+1♀(2sp) was around 10 ( Fig. 1h) but, that of 1♂+2♀ 368 (2sp) was around 15-20 (Supplementary Figure 5), indicating that the lower ability to decrease 369 histone mobility of fPN. 370 Furthermore, in figure 1i, we compared ROSI, ELSI, tICSI and ICSI-zygotes. The 371 results showed that histone mobility in female PN was drastically changed depending on their 372 partner male germ cells. In contrast, rs-mPN and sp-mPN showed no significant changes in 373 histone mobility despite their differences in resistance to the ability for decreasing histone 374 mobility (Supplementary Figure 9, 10). 375 Collectively these results indicated that if there are chromatin compaction factors 376 derived from fPN, it would have little impact on parental asymmetry. Therefore, we modified 377 and added the sentence below to the result section. Furthermore, fPN in ROSI-zygotes showed higher histone mobility than ICSI-zygotes, 387

Response to reviewers' comments
indicating the scarce or little repressive effect on histone mobility by fPN. 388 (p50 line 788-795) 414 "(a) During spermiogenesis, which is the post-meiotic stage, male germ cells change the 415 morphology dynamically and differentiate from round spermatid (RS) to elongating spermatid 416 (ES) and testicular sperm (tSP) in the testis. After differentiation to tSP, they move to cauda 417 epididymis from the testis and then mature to sperm (SP). Micro-insemination with this SP is 418 called ICSI, and with round spermatid, elongating spermatid in the testis is as ROSI and ELSI 419 (elongating spermatid injection), respectively. We referred to microinjection with immature tSP 420 as "testicular ICSI" (tICSI)." 421 422 To explain the purpose of the experiment with inactivated sperm, we added the explanation 423 sentence below to the result section and a simple illustration (Supplementary Figure 6f). The 2 figures below are cited from our previous study (Ooga et al. 2016).